Long-chain .alpha., .omega.-alkanedicarboxylic acids (i.e., alkanedicarboxylic acids with a carbon number of nine or greater) are used as raw materials in the synthesis of a variety of chemical products and polymer materials. For example, dodecanedioic acid is used as a comonomer with bisphenol A to produce a copolyestercarbonate, a particularly desirable engineering thermoplastic which retains high impact strength, the hallmark of polycarbonate resin, while having a lower melt viscosity. This property results in better molding productivity than conventional polycarbonate resin, allowing the production of light, strong, thin-walled plastic parts.
Diacids with carbon numbers greater than four (hereinafter referred to as diacids) are currently produced almost exclusively by nonbiological conversion processes. Dodecanedioic acid is manufactured through the nickel-catalyzed cyclic trimerization of butadiene, followed by hydrogenation to cyclododecane, air oxidation to a mixture of cyclododecanone and cyclododecanol, and finally, nitric acid oxidation to dodecanedioic acid. These types of chemical processes for the production of diacids have a number of limitations and disadvantages. Each process is restricted to the production of diacids of specific carbon chain lengths, based on the starting material used. For example, the dodecanedioic acid process begins with butadiene, therefore the products of this reaction process are limited to acids with chain lengths in multiples of four. In practice, only a single diacid, dodecanedioic acid, is made by this process. In addition, the processes are based on nonrenewable petrochemical feedstocks, and the multireaction conversion process produces unwanted byproducts which result in yield loses, heavy metal wastes, and nitrogen oxides which must be destroyed in a reduction furnace.
Biological conversion processes for the production of diacids have a number of potential advantages relative to the existing non-biological conversion processes. Primary among these is the use of renewable feedstocks as starting materials and the ability to produce the diacid without the generation of hazardous chemical byproducts which necessitate costly waste disposal processes. Another important advantage achieved by using a biological process is that such a process can easily be adapted to produce a wide variety of diacids using the same biocatalyst and the same equipment. Because current organic chemical syntheses are suited to the production of only a single diacid, the synthesis of several different diacids would require the development of a new synthetic scheme for each diacid. On the other hand, a yeast biocatalyst can be used to produce diacids of varying lengths using the same equipment, media and protocols merely by providing a different substrate to the yeast.
Several naturally occurring yeasts are known to produce diacids when provided fatty acids, fatty acid esters or alkanes as substrates. Yeasts belonging to the genus Candida, such as C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. lipolytica, C. maltosa, C. parapsilosis, C. zeylenoides, and C. tropicalis have been reported to produce diacids. These yeasts have a number of limitations preventing their use for the commercial production of diacids, however. They produce an inefficient total yield of diacid relative to the fatty acid or alkane starting material, and they also produce relatively large quantities of unwanted byproducts.
In yeast, n-alkane substrates are transported into the cell, hydroxylated to fatty alcohols by a specific cytochrome P450 system, and then further oxidized by an alcohol oxidase and an aldehyde oxidase to form a fatty acid. Fatty acids are oxidized in the same way to form the corresponding diacid. Both fatty acids and diacids can be degraded, however, through the peroxisomal .quadrature.-oxidation pathway subsequent to activation to an acyl-CoA ester. This leads to shortening of the chain by units of two. Thus, diacids produced in yeasts are often shortened to differing degrees.
Genetically modified strains of Candida tropicalis have been developed that overcome some of these obstacles to cost-effective commercial production of diacids. U.S. Pat. No. 5,254,466 discloses the genetic modification of a C. tropicalis strain. In this strain of yeast, the beta-oxidation pathway is blocked, resulting in the production of substantially pure diacid without the unwanted conversion of the substrate into shorter chain diacids and biomass, and in substantially quantitative yield with respect to the starting material. A particular organism blocked for .quadrature.-oxidation is C. tropicalis H5343 (ATCC No. 20962).
C. tropicalis has been further modified, as described in U.S. Pat. No. 5,620,878. This reference discloses amplification of the genes encoding cytochrome P450 monooxygenase and NADPH reductase, which results in increased 4-hydroxylase activity and thus increased specific productivity (grams of diacid/liter/hr) of diacids. The particular organism having amplified cytochrome P450 monooxygenase and NADPH reductase components is known as C. tropicalis AR40 (ATCC No. 20987).
The known genetically modified strains show distinct advantages over previously available yeast strains for the production of diacids. This technology has resulted in organisms which are able to produce the large quantities of product necessary to develop a commercially feasible process. The described production using these prior art methods, however, includes the use of preferred fermentation media which contain relatively expensive components. The cost of the medium in the biofermentation processes is too high for practical use in diacid production on a large commercial scale. For example, the preferred embodiment of U.S. Pat. No. 5,620,878 describes a medium comprising 3 g/L peptone, 6 g/L yeast extract, 6.7 g/L Yeast Nitrogen Base (Difco), 3 g/L sodium acetate, 7.2 g/L K.sub.2 HPO.sub.4 .multidot.3H.sub.2 O, 9.3 g/L KH.sub.2 PO.sub.4, and 75 g/L glucose. These are ingredients suitable for laboratory practice, but not industrial scale fermentation.
Consequently, there remains a need for low-cost, complete biofermentation media which provide sufficient nutrient support to the yeast biocatalyst to permit high specific productivity of diacids from suitable fatty acid or alkane starting material.